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BIPN140 Lecture 7: Synaptic Transmission I 1. Electrical vs. Chemical Synapses 2. Neurotransmitters (NTs) 3. Presynaptic Machinery (NT release) Annotated Slides, Su (FA15) Propagation of Information between Neurons Pre-synaptic cell Post-synaptic cell Synapse Synapses: Specific contact between a neuron and another cell (neuron or effector cell) for signal transmission Feature/Functions: 1. Local transmission: specific wiring diagram (physical connections. 2. Temporal control: can be rapid or longer lasting 3. Synaptic plasticity: encode changes (e.g. learning & memory) Challenges: 1. Molecular machinery 2. Target selection 3. Synaptic plasticity

BIPN140 Lecture 7: Synaptic Transmission Iclasses.biology.ucsd.edu/bipn140.FA16/BIPN140_Handout7_Su.pdf · Synaptic Transmission: (1) Action potentials in presynaptic nerve terminals

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Page 1: BIPN140 Lecture 7: Synaptic Transmission Iclasses.biology.ucsd.edu/bipn140.FA16/BIPN140_Handout7_Su.pdf · Synaptic Transmission: (1) Action potentials in presynaptic nerve terminals

BIPN140 Lecture 7: Synaptic Transmission I

1. Electrical vs. Chemical Synapses

2. Neurotransmitters (NTs)

3. Presynaptic Machinery (NT release)

Annotated Slides, Su (FA15)

Propagation of Information between Neurons

Conduct messagesPre-synaptic cell

Post-synaptic cell

Synapse

Synapses: Specific contact between a neuron and another cell (neuron or effector cell) for signal transmission

Feature/Functions:1. Local transmission: specific wiring diagram (physical

connections.2. Temporal control: can be rapid or longer lasting3. Synaptic plasticity: encode changes (e.g. learning &

memory)

Challenges:1. Molecular machinery2. Target selection3. Synaptic plasticity

Page 2: BIPN140 Lecture 7: Synaptic Transmission Iclasses.biology.ucsd.edu/bipn140.FA16/BIPN140_Handout7_Su.pdf · Synaptic Transmission: (1) Action potentials in presynaptic nerve terminals

Two Types of Synapses: Electrical & Chemical

Current flow directly from one neuron to another via gap junction.

One neuron communicates with another via the secretion of neurotransmitters.

Electrical Synapse/Gap Junction (Fig. 5.1)

Structure:

(1) Gap between pre- & post-

synaptic membrane.

(2) Connexons: channels comprising membrane pores (six connexin subunits), present at both pre- and post-synaptic membrane.

Function:

(1) Direct charge transfer (passive).

(2) Exchange of 2nd messengers &

intracellular metabolites to

coordinate intracellular signaling.

(3) Rapid transmission w/o delay.

(4) Synchronized electrical activity.

Features:

(1) Distinct minority but found in

all nervous systems.

(2) Could allow bidirectional

transmission.

Gap Junction: Intercellular specialization containing aligned, paired channels called connexons or hemichannels (channels with a much larger pore).

Page 3: BIPN140 Lecture 7: Synaptic Transmission Iclasses.biology.ucsd.edu/bipn140.FA16/BIPN140_Handout7_Su.pdf · Synaptic Transmission: (1) Action potentials in presynaptic nerve terminals

Electrical Synapse (Fig. 5.2)

Crayfish nervous system Hippocampal interneurons

Rapid transmission Synchronized firing of action potentials

1. Brainstem neurons2. Interneurons in the cortex, thalamus,

cerebellum, hippocampus and other regions.3. Hormone-secreting cells in the hypothalamus4. Between glia cells (exchange of ions and

metabolites, forming a signaling network)

Chemical Synapse: an Overview (Fig. 5.3)

Page 4: BIPN140 Lecture 7: Synaptic Transmission Iclasses.biology.ucsd.edu/bipn140.FA16/BIPN140_Handout7_Su.pdf · Synaptic Transmission: (1) Action potentials in presynaptic nerve terminals

Chemical Synapse

Structure: (1) Presynaptic terminal:

a. voltage-sensitive Ca2+ channels

b. synaptic vesicles containing neurotransmitter

(2) Postsynaptic dendrites:

concentrated neurotransmitter receptors and scaffold proteins

Synaptic Transmission:

(1) Action potentials in presynaptic nerve terminals induce

exocytosis of synaptic vesicles to release neurotransmitters.

(2) Neurotransmitters diffuse rapidly across synaptic cleft (very

narrow gap, <50 nm, to allow short diffusion time).

(3) Neurotransmitters bind to and activate neurotransmitter

receptors on postsynaptic membrane to open the channels

(ligand-gated ion channels or “ionotropic receptors”) or trigger

intracellular signaling events (metabotropic receptors).

(4) Ions flow through the open receptor channels to produce

postsynaptic potential (graded potential in the postsynaptic

neuron)

Major Neurotransmitters

Many types of neurotransmitters (>100)

A given neurotransmitter can bind to different types of receptor.

Therefore, a given neurotransmitter can excite postsynaptic cells expressing one receptor and inhibit others expressing a different receptor. The effect of a given neurotransmitter is determined by the receptor it activates.

Page 5: BIPN140 Lecture 7: Synaptic Transmission Iclasses.biology.ucsd.edu/bipn140.FA16/BIPN140_Handout7_Su.pdf · Synaptic Transmission: (1) Action potentials in presynaptic nerve terminals

Criteria of Neurotransmitters at a Given Synapse (Box 5A)

(1) Location: present in the synaptic terminal

(2) Release is triggered by presynaptic depolarization elicited Ca2+ influx

(3) Response: when applied directly to postsynaptic cells, it

produces postsynaptic potentials (PSP) or intracellular signaling events.

That is, postsynaptic cells have appropriate receptors for the NT.

Metabolism of Neurotransmitters (Fig. 5.5)

Small NTs:

(1) For example, acetylcholine, glutamate, GABA etc.

(2) Synthesized locally at the presynaptic terminal (precursors are supplied locally)

(3) Recycling: reuptake or enzymatic inactivation

(4) Abundant (numerous synaptic vesicles)

(5) Small clear-core vesicles (SCCVs, 40-60 nm)

(6) Slow axonal transport of the enzymes (0.5-5.0 mm/day)

Large NTs:

(1) Neuropeptides, biogenic amines

(2) Synthesized mainly at the cell body, rely on axonal transport

(3) Degraded by proteolytic enzymes

(4) Less abundant (fewer vesicles)

(5) Large dense-core vesicles (LDCVs, 90-250 nm)

(6) Fast axonal transport of the NTs and enzymes (400 mm/day, 80-800x faster)

Page 6: BIPN140 Lecture 7: Synaptic Transmission Iclasses.biology.ucsd.edu/bipn140.FA16/BIPN140_Handout7_Su.pdf · Synaptic Transmission: (1) Action potentials in presynaptic nerve terminals

Differential Release of NTs (Fig. 5.12)

(1) It is common that a neuron can release multiple types of NTs (co-transmitters).

(2) Low-frequency stimulation preferentially raises the Ca2+

concentration close to the membrane, favoring the release of SCCVs (small-molecule NTs).

(3) High-frequency stimulation leads to a more general increase of Ca2+ concentration, causing the release of both LDCVs (neuropeptides) and SCCVs (small-molecule NTs).

Synaptic Transmission at the Neuromuscular Junction (Fig. 5.6)

Neuromuscular Junction (NMJ):

(1) Experimental paradigm for chemical synaptic transmission: simple, large and accessible.

(2) Presynaptic neuron releases ACh, postsynaptic cell expresses AChR.

(3) Specialized synaptic structure: end plates

(4) End plate potential (EPP): intracellular recording of the membrane potential at the postsynaptic muscle cell.

(5) Pronounced delay (milliseconds) between stimulus and response, characteristics of chemical synapses.

Delay between stimulus and EPP responses

spinal motor

neuron

Page 7: BIPN140 Lecture 7: Synaptic Transmission Iclasses.biology.ucsd.edu/bipn140.FA16/BIPN140_Handout7_Su.pdf · Synaptic Transmission: (1) Action potentials in presynaptic nerve terminals

Spontaneous miniature EPPs and evoked EPPs (Fig. 5.6)

Spontaneous miniature EPPs (MEPPs):

(1) Occurs spontaneously without presynaptic stimulation.

(2) Very small (~1 mV), as opposed to evoked EPPs (>50 mV), likely due to fewer SVs release.

(3) Blocked by AChR inhibitor (e.g. curare).

Evoked EPPs:

(1) Magnitude is much reduced in low Ca2+

extracellular solution (reduce the number of SVs being released, or Mg2+ rich solution).

(2) Subthreshold EPPs to prevent action potential and muscle contraction.

(3) Analysis of subthreshold EPPs gives insights into the mechanisms of NT release.

(4) Unitary EPP = smallest MEPP

(5) How about blocking nAChR with curare?

Quantal Release of Neurotransmitters (Fig. 5.7)

“Unit” amounts of ACh release:

(1) Poisson Statistics: based on the independent occurrence of unitary events (assuming that EPPs are built up from unitary events represented by MEPPs).

(2) Increments of the EPP response occur in units about the size of single MEPP.

(3) Poisson distribution: discrete probability distribution

(4) ACh is released in discrete packets

Low extracellular Ca2+

Peaks of EPP amplitudes tend to occur in integer multiples of the mean amplitude of MEPPs.

Page 8: BIPN140 Lecture 7: Synaptic Transmission Iclasses.biology.ucsd.edu/bipn140.FA16/BIPN140_Handout7_Su.pdf · Synaptic Transmission: (1) Action potentials in presynaptic nerve terminals

NT Quantal Release: Synaptic Vesicle Exocytosis (Fig. 5.8)

Quantal SV release:

(1) EM: SVs at the presynaptic terminals

(2) SVs: the source of the quanta, repositories of neurotransmitters (~10,000 ACh molecules/SV)

(3) MEPP: discharge of the contents of single SVs

(4) m = n x p

m = mean number of quanta (EPPs or PSPs/MEPP)n = total number of quanta (SVs) available

for release p = probability of releasing a quantum (SV)

4-AP: 4-aminopyridine, drug that blocks voltage-gated K+ channels to enhance the number of quantal release. Why? Lengthening the duration of presynaptic AP.

Readily releasable SVs

Voltage-gated calcium channels (VGCCs)

Measured by EPP/MEPP

Measured by EM

Membrane capacitance ~1 F/cm2 (10-14 Farad/m2)

Vesicle surface area: assuming the diameter is 50 nm (0.05 m), 4r2 = 0.008 m2

One quantal release: 0.008 x 10-14 Farad/m2 = 8 x10-17 FaradIf 5000 SVs are released: 4 x10-13 increase in total membrane capacitance, ~0.5% increase (8 x 10-11 Farad)

Local Recycling of SVs (Fig. 5.9)

DAB staining for EM(or fluorescent dye for live imaging)

Page 9: BIPN140 Lecture 7: Synaptic Transmission Iclasses.biology.ucsd.edu/bipn140.FA16/BIPN140_Handout7_Su.pdf · Synaptic Transmission: (1) Action potentials in presynaptic nerve terminals

Presynaptic Vesicle Trafficking (Fig. 5.13B)

Vesicle Trafficking Steps:

Reserve pool => mobilization (phosphorylation of synapsin) => docking/priming => fusion (NT release) => recovery of SV by endocytosis (clathrin coating, budding, un-coating) => reloading of NT by vesicular neurotransmitter transporters

Proteins on SVs: v-SNAREs (Fig. 5.13A)

Vesicle SNAREs (v-SNAREs):

(1) The precise mechanism for SV trafficking is still not clear.

(2) A large number of proteins are found on SVs. They mediate different steps of SVs trafficking.

(3) Synapsin: retains SVs in the reserve pool, links to actin. CaMKII (calcium-calmodulin dependent protein kinase II): regulates synapsin to release SVs from actin (mobilization)

(4) Many proteins are involved in priming, a process to organize SNARE proteins into the correct conformation for membrane fusion.

(5) NT transporters such as VGLUTs(vesicular glutamate transporters)

Page 10: BIPN140 Lecture 7: Synaptic Transmission Iclasses.biology.ucsd.edu/bipn140.FA16/BIPN140_Handout7_Su.pdf · Synaptic Transmission: (1) Action potentials in presynaptic nerve terminals

Exocytosis during Synaptic Release (Fig. 5.14)

Target SNAREs (t-SNAREs):

(1) Proteins located in the membranes of target compartments

(2) Key t-SNAREs for SV docking: syntaxin & SNAP-25 (target of Botulinum toxin, a.k.a. Botox).

Fusion Events:

(1) v- and t-SNAREs form tight macro-molecular complex to pull membranes together. Key v-SNAREs: synaptobrevin. (target of TeNT & some Botox)

(2) Ca2+ enters and binds to synaptotagmin, leading to membrane fusion to permit NT release.

myasthenic syndromes:Loss of VDCCs? Loss of ACh receptors? Loss of acetylcholinesterase?(presynaptic vs postsynaptic deficit)

C2 domain

Endocytosis following Synaptic Release (Fig. 5.15)

Endocytosis Events:

(1) Recycling of SVs driven by clathrin coating (curving) and dynamin excision (pinching off).

(2) Extra membrane is removed within a few minutes.

(3) Importance:

a) presynaptic membrane homeostasis (capacitance)b) replenishing SVs locally is much faster (otherwise it is

too slow to transport SVs from ER/Golgi apparatus at soma).

Initiate membrane budding

ATPase removes clathrin coats

Page 11: BIPN140 Lecture 7: Synaptic Transmission Iclasses.biology.ucsd.edu/bipn140.FA16/BIPN140_Handout7_Su.pdf · Synaptic Transmission: (1) Action potentials in presynaptic nerve terminals

SVs distribute between reserve pool and readily-releasable pool (docked and primed) which have fully formed SNARE complexes.

Local Ca2+ entry (AP transiently opens voltage-gated calcium channels) alters synaptotagmin conformation, which in turn unleashes the SNARE complex in a way that allows sufficient fusion with the plasma membrane to form a pore through which the NTs quickly diffuse into the synaptic cleft.

Result: producing a “quantal” NT release event.

The fused vesicle membrane is recycled soon afterwards to replenish SVs at the presynaptic terminal.

Summary

Question: which molecule(s) functions as the key “calcium sensor” for SV release?

What is known: the latency between Ca2+ entry and SV release is very short (200 s), suggesting (1) that SVs are “ready to go” (docked and primed); (2) Ca2+ sensor is in close proximity to SVs.

Page 12: BIPN140 Lecture 7: Synaptic Transmission Iclasses.biology.ucsd.edu/bipn140.FA16/BIPN140_Handout7_Su.pdf · Synaptic Transmission: (1) Action potentials in presynaptic nerve terminals

Synaptotagmin (SYTs)

Localized in the membrane of SVs.

Contains domains that allow Ca2+-dependent binding of the protein to phospholipid membrane.

Hypothesis: (1) synaptotagmin is a Ca2+ binding protein at physiological Ca2+ concentrations.

(2) Ca2+-bound synaptotagmin interacts with phospholipid membrane

synaptotagmin phospholipid vesicle

phospholipid vesicle

synaptotagmin

Tryptophan (in synaptotagmin) & dansyl

group in the liposome, energy transfer depends on the distance between

the two molecules

Reversible interaction